Colonization and Competition Dynamics of Plant Growth-Promoting/Inhibiting Bacteria in the Phytosphere of the Duckweed Lemna minor

  • Hidehiro Ishizawa
  • Masashi Kuroda
  • Kanako Inoue
  • Daisuke Inoue
  • Masaaki Morikawa
  • Michihiko IkeEmail author
Plant Microbe Interactions


Despite the considerable role of aquatic plant-associated bacteria in host plant growth and nutrient cycling in aquatic environments, the mode of their plant colonization has hardly been understood. This study examined the colonization and competition dynamics of a plant growth-promoting bacterium (PGPB) and two plant growth-inhibiting bacteria (PGIB) in the aquatic plant Lemna minor (common duckweed). When inoculated separately to L. minor, each bacterial strain quickly colonized at approximately 106 cells per milligram (plant fresh weight) and kept similar populations throughout the 7-day cultivation time. The results of two-membered co-inoculation assays revealed that the PGPB strain Aquitalea magnusonii H3 consistently competitively excluded the PGIB strain Acinetobacter ursingii M3, and strain H3 co-existed at almost 1:1 proportion with another PGIB strain, Asticcacaulis excentricus M6, regardless of the inoculation ratios (99:1–1:99) and inoculation order. We also found that A. magnusonii H3 exerted its growth-promoting effect over the negative effects of the two PGIB strains even when only a small amount was inoculated, probably due to its excellent competitive colonization ability. These experimental results demonstrate that there is a constant ecological equilibrium state involved in the bacterial colonization of aquatic plants.


Duckweed Plant growth-promoting bacteria Plant growth-inhibiting bacteria Colonization Competition Aquatic plant 



We thank Dr. Takao Sakata and Ms. Minami Tada for their support in experiments.

Funding Information

This work was supported by the Advanced Low Carbon Technology Research and Development Program Grant Number JPMJAL1108 of the Japan Science and Technology Agency and the Japan Society for the Promotion of Science KAKENHI Grant Number JP18J10181. A part of this work was supported by the Advanced Characterization Nanotechnology Platform, Nanotechnology Platform Program of the Ministry of Education, Culture, Sports, Science and Technology, Japan, at the Research Center for Ultra-High Voltage Electron Microscopy (Nanotechnology Open Facilities) in Osaka University.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

Ethical Approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

248_2018_1306_MOESM1_ESM.docx (16 kb)
Supplementary Table 1 (DOCX 16 kb)
248_2018_1306_MOESM2_ESM.xlsx (13 kb)
Supplementary Table 2 (XLSX 13 kb)


  1. 1.
    Rezania S, Ponraj M, Din MFM, Songip AR, Sairan FM, Chelliapan S (2012) The diverse applications of water hyacinth with main focus on sustainable energy and production for new era: an overview. Renew Sust Energ Rev 41:943–954. CrossRefGoogle Scholar
  2. 2.
    Ziegler P, Sree S, Appenroth KJ (2016) Duckweeds for water remediation and toxicity testing. Toxicol Environ Chem 98:1127–1154. CrossRefGoogle Scholar
  3. 3.
    Bayrakci AG, Koçar G (2014) Second-generation bioethanol production from water hyacinth and duckweed in Izmir: a case study. Renew Energ Sust Rev 30:306–316. CrossRefGoogle Scholar
  4. 4.
    Cui W, Cheng JJ (2015) Growing duckweed for biofuel production: a review. Plant Biol 17:16–23. CrossRefPubMedGoogle Scholar
  5. 5.
    Cheng JJ, Stomp AM (2009) Growing duckweed to recover nutrients from wastewaters and for production of fuel ethanol and animal feed. Clean (Weinh) 37:17–26. CrossRefGoogle Scholar
  6. 6.
    Rezania S, Taib SM, Din MFM et al (2016) Comprehensive review on phytotechnology: heavy metals removal by diverse aquatic plants species from wastewater. J Hazard Mater 318:587–599. CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Fujita M, Mori K, Kodera T (1999) Nutrient removal and starch production through cultivation of Wolffia arrhiza. J Biosci Bioeng 87:194–198. CrossRefPubMedGoogle Scholar
  8. 8.
    Mishima D, Kuniki M, Sei K, Soda S, Ike M, Fujita M (2008) Ethanol production from candidate energy crops: water hyacinth (Eichhornia crassipes) and water lettuce (Pistia stratiotes L.). Bioresour Technol 99:2495–2500. CrossRefPubMedGoogle Scholar
  9. 9.
    Ge X, Zhang N, Phillips GC, Xu J (2012) Growing Lemna minor in agricultural wastewater and converting the duckweed biomass to ethanol. Bioresour Technol 124:485–488. CrossRefPubMedGoogle Scholar
  10. 10.
    Mohedano RA, Costa RHR, Tavares FA, Belli Filho P (2012) High nutrient removal rate from swine wastes and protein biomass production by full-scale duckweed ponds. Bioresour Technol 112:98–104. CrossRefPubMedGoogle Scholar
  11. 11.
    Zhao Y, Fang Y, Jin Y, Huang J, Bao S, Fu T, He Z, Wang F, Zhao H (2014) Potential of duckweed in the conversion of wastewater nutrients to valuable biomass: a pilot-scale comparison with water hyacinth. Bioresour Technol 163:82–91. CrossRefPubMedGoogle Scholar
  12. 12.
    Soda S, Ohchi T, Piradee J, Takai Y, Ike M (2015) Duckweed biomass as a renewable biorefinery feedstock: ethanol and succinate production from Wolffia globosa. Biomass Bioenergy 81:364–368. CrossRefGoogle Scholar
  13. 13.
    Toyama T, Hanaoka T, Tanaka Y, Morikawa M, Mori K (2018) Comprehensive evaluation of nitrogen removal rate and biomass, ethanol, and methane production yields by combination of four major duckweeds and three types of wastewater effluent. Bioresour Technol 250:464–473. CrossRefPubMedGoogle Scholar
  14. 14.
    Underwood GJC, Baker JH (1991) The effect of various aquatic bacteria on the growth and senescence of duckweed (Lemna minor). J Appl Bacteriol 70:192–196. CrossRefGoogle Scholar
  15. 15.
    Ishizawa H, Kuroda M, Morikawa M, Ike M (2017) Evaluation of environmental bacterial communities as a factor affecting the growth of duckweed Lemna minor. Biotechnol Biofuels 10(62):62. CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Stout L, Nüsslein K (2010) Biotechnological potential of aquatic plant-microbe interactions. Curr Opin Biotechnol 21:339–345. CrossRefPubMedGoogle Scholar
  17. 17.
    Wei B, Yu X, Zhang S, Gu L (2011) Comparison of the community structures of ammonia-oxidizing bacteria and archaea in rhizoplanes of floating aquatic macrophytes. Microbiol Res 166:468–474. CrossRefPubMedGoogle Scholar
  18. 18.
    Tsuji K, Asayama T, Shiraki N, Inoue S, Okuda E, Hayashi C, Nishida K, Hasegawa H, Harada E (2017) Mn accumulation in a submerged plant Egeria densa (Hydrocharitaceae) is mediated by epiphytic bacteria. Plant Cell Environ 40:1163–1173. CrossRefPubMedGoogle Scholar
  19. 19.
    Zhao Y, Fang Y, Jin Y, Huang J, Ma X, He K, He Z, Wang F, Zhao H (2015) Microbial community and removal of nitrogen via the addition of a carrier in a pilot-scale duckweed-based wastewater treatment system. Bioresour Technol 179:549–558. CrossRefPubMedGoogle Scholar
  20. 20.
    Toyama T, Furukawa T, Maeda N, Inoue D, Sei K, Mori K, Kikuchi S, Ike M (2011) Accelerated biodegradation of pyrene and benzo [a] pyrene in the Phragmites australis rhizosphere by bacteria-root exudate interactions. Water Res 45:1629–1638. CrossRefPubMedGoogle Scholar
  21. 21.
    Chen W, Tang Y, Mori K, Wu XL (2012) Distribution of culturable endophytic bacteria in aquatic plants and their potential for bioremediation in polluted waters. Aquat Biol 15:99–110. CrossRefGoogle Scholar
  22. 22.
    Yamaga F, Washio K, Morikawa M (2010) Sustainable biodegradation of phenol by Acinetobacter calcoaceticus P23 isolated from the rhizosphere of duckweed Lemna aoukikusa. Environ Sci Technol 44:6470–6474. CrossRefPubMedGoogle Scholar
  23. 23.
    El-Deeb B, Gherbawy Y, Hassan S (2012) Molecular characterization of endophytic bacteria from metal hyperaccumulator aquatic plant (Eichhornia crassipes) and its role in heavy metal removal. Geomicrobiol J 29:906–915. CrossRefGoogle Scholar
  24. 24.
    Ogata Y, Goda S, Toyama T, Sei K, Ike M (2013) The 4-tert-butylphenol-utilizing bacterium Sphingobium fuliginis OMI can degrade bisphenols via phenolic ring hydroxylation and meta-cleavage pathway. Environ Sci Technol 47:1017–1023. CrossRefPubMedGoogle Scholar
  25. 25.
    Tang J, Zhang Y, Cui Y, Ma J (2009) Effects of a rhizobacterium on the growth of and chromium remediation by Lemna minor. Environ Sci Pollut Res 22:9686–9693. CrossRefGoogle Scholar
  26. 26.
    Lugtenberg B, Kamilova F (2009) Plant-growth-promoting rhizobacteria. Annu Rev Microbiol 63:541–556. CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Faisal M, Hasnain S (2005) Beneficial role of hydrophytes in removing Cr (VI) from wastewater in association with chromate-reducing bacterial strains Ochrobactrum intermedium and Brevibacterium. Int J Phytoremediation 7:271–277. CrossRefPubMedGoogle Scholar
  28. 28.
    Kristanti RA, Toyama T, Hadibarata T, Tanaka Y, Mori K (2014) Bioaugmentation involving a bacterial consortium isolated from the rhizosphere of Spirodela polyrhiza for treating water contaminated with a mixture of four nitrophenol isomers. RSC Adv 4:1616–1621. CrossRefGoogle Scholar
  29. 29.
    Xu XJ, Sun JQ, Nie Y, Wu XL (2015) Spirodela polyrhiza stimulates the growth of its endophytes but differentially increases their fenpropathrin-degradation capabilities. Chemosphere 125:33–40. CrossRefPubMedGoogle Scholar
  30. 30.
    Toyama T, Kuroda M, Ogata Y, Hachiya Y, Quach A, Tokura K, Tanaka Y, Mori K, Morikawa M, Ike M (2017) Enhanced biomass production of duckweeds by inoculating a plant growth-promoting bacterium, Acinetobacter calcoaceticus P23, in sterile medium and non-sterile environmental waters. Water Sci Technol 76:1418–1428. CrossRefPubMedGoogle Scholar
  31. 31.
    Whipps JM (2001) Microbial interactions and biocontrol in the rhizosphere. J Exp Bot 52:487–511. CrossRefPubMedGoogle Scholar
  32. 32.
    Raaijmakers JM, Mazzola M (2012) Diversity and natural functions of antibiotics produced by beneficial and plant pathogenic bacteria. Annu Rev Phytopathol 50:403–424. CrossRefPubMedGoogle Scholar
  33. 33.
    Lugtenberg B, Dekkers L, Bloemberg GV (2001) Molecular determinants of rhizosphere colonization by Pseudomonas. Annu Rev Phytopathol 9:461–490. CrossRefGoogle Scholar
  34. 34.
    Liu W, Yang C, Shi S, Shu W (2014) Effects of plant growth-promoting bacteria isolated from copper tailings on plants in sterilized and non-sterilized tailings. Chemosphere 97:47–53. CrossRefPubMedGoogle Scholar
  35. 35.
    Suzuki W, Sugawara M, Miwa K, Morikawa M (2014) Plant growth-promoting bacterium Acinetobacter calcoaceticus P23 increases the chlorophyll content of the monocot Lemna minor (duckweed) and the dicot Lactuca sativa (lettuce). J Biosci Bioeng 118:41–44. CrossRefPubMedGoogle Scholar
  36. 36.
    Toyama T, Yu N, Kumada H, Sei K, Ike M, Fujita M (2006) Accelerated aromatic compounds degradation in aquatic environment by use of interaction between Spirodela polyrrhiza and bacteria in its rhizosphere. J Biosci Bioeng 101:346–353. CrossRefPubMedGoogle Scholar
  37. 37.
    Ishizawa H, Kuroda M, Morikawa M (2017) Differential oxidative and antioxidative response of duckweed Lemna minor toward plant growth promoting/inhibiting bacteria. Plant Physiol Biochem 118:667–673. CrossRefPubMedGoogle Scholar
  38. 38.
    Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8:4321–4325. CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Pedersen K (1982) Method for studying microbial biofilms in flowing-water systems. Appl Environ Microbiol 43:6–13PubMedPubMedCentralGoogle Scholar
  40. 40.
    Lardi M, de Campos SB, Purtschert G, Eberl L, Pessi G (2017) Competition experiments for legume infection identify Burkholderia phymatum as a highly competitive β-rhizobium. Front Microbiol 8(1527).
  41. 41.
    Xiong H, Li Y, Cai Y, Cao Y, Wang Y (2015) Isolation of Bacillus amyloliquefaciens JK6 and identification of its lipopeptides surfactin for suppressing tomato bacterial wilt. RSC Adv 5:82042–82049. CrossRefGoogle Scholar
  42. 42.
    Ofek M, Voronov-Goldman M, Hadar Y, Minz D (2014) Host signature effect on plant root-associated microbiomes revealed through analyses of resident vs. active communities. Environ Microbiol 16:2157–2167. CrossRefPubMedGoogle Scholar
  43. 43.
    Xie WY, Su JQ, Zhu YG (2015) Phyllosphere bacterial community of floating macrophytes in paddy soil environments as revealed by Illumina high-throughput sequencing. Appl Environ Microbiol 81:522–532. CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Hamonts K, Trivedi P, Garg A, Janitz C, Grinyer J, Holford P, Botha FC, Anderson IC, Singh BK (2018) Field study reveals core plant microbiota and relative importance of their drivers. Environ Microbiol 20:124–140. CrossRefPubMedGoogle Scholar
  45. 45.
    Barret M, Morrissey JP, O’Gara F (2011) Functional genomics analysis of plant growth-promoting rhizobacterial traits involved in rhizosphere competence. Biol Fertil Soils 47:729–743. CrossRefGoogle Scholar
  46. 46.
    Hatzinger PB, Alexander M (1994) Relationship between the number of bacteria added to soil or seeds and their abundance and distribution in the rhizosphere of alfalfa. Plant Soil 158:211–222. CrossRefGoogle Scholar
  47. 47.
    Normander B, Prosser JI (2000) Bacterial origin and community composition in the barley phytosphere as a function of habitat and presowing conditions. Appl Environ Microbiol 66:4372–4377. CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Adame-Álvarez RM, Mendiola-Soto J, Heil M (2014) Order of arrival shifts endophyte-pathogen interactions in bean from resistance induction to disease facilitation. FEMS Microbiol Lett 355:100–107. CrossRefPubMedGoogle Scholar
  49. 49.
    Chesson P (1994) Multispecies competition in variable environments. Theor Popul Biol 45:227–276. CrossRefGoogle Scholar
  50. 50.
    Matsuzawa H, Tanaka Y, Tamaki H, Kamagata Y, Mori K (2010) Culture-dependent and independent analyses of the microbial communities inhabiting the giant duckweed (Spirodela polyrrhiza) rhizoplane and isolation of a variety of rarely cultivated organisms within the phylum Verrucomicrobia. Microbes Environ 25:302–308. CrossRefPubMedGoogle Scholar
  51. 51.
    Quisehuatl-Tepexicuapan E, Ferrera-Cerrato R, Silva-Rojas HV et al (2016) Free-living culturable bacteria and protozoa from the rhizoplanes of three floating aquatic plant species. Plant Biosyst 150:855–865. CrossRefGoogle Scholar
  52. 52.
    De-Bellis P, Ercolani GL (2001) Growth interactions during bacterial colonization of seedling rootlet. Appl Environ Microbiol 67:1945–1948. CrossRefPubMedPubMedCentralGoogle Scholar
  53. 53.
    Levine JM, HilleRisLambers J (2009) The importance of niches for the maintenance of species diversity. Nature 461:254–257. CrossRefPubMedGoogle Scholar
  54. 54.
    Ishizawa H, Kuroda M, Ike M (2017) Draft genome sequence of Aquitalea magnusonii strain H3, a plant growth-promoting bacterium of duckweed (Lemna minor). Genome Announc 5:e00812–e00817. CrossRefPubMedPubMedCentralGoogle Scholar
  55. 55.
    Singh M, Awasthi A, Soni SK, Singh R, Verma RK, Kalra A (2015) Complementarity among plant growth promoting traits in rhizospheric bacterial communities promotes plant growth. Sci Rep 5:15500. CrossRefPubMedPubMedCentralGoogle Scholar
  56. 56.
    Fan B, Borriss R, Bleiss W, Wu X (2012) Gram-positive rhizobacterium Bacillus amyloliquefaciens FZB42 colonizes three types of plants in different patterns. J Microbiol 50:38–44. CrossRefPubMedGoogle Scholar
  57. 57.
    Appenroth KJ, Ziegler P, Sree S (2017) Duckweed as a model organism for investigating plant-microbe interactions in an aquatic environment and its applications. Endocytobiosis Cell Res 27:94–106Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Division of Sustainable Energy and Environmental Engineering, Graduate School of EngineeringOsaka UniversityOsakaJapan
  2. 2.Research Center for Ultra-High Voltage Electron MicroscopyOsaka UniversityOsakaJapan
  3. 3.Division of Biosphere Science, Graduate School of Environmental ScienceHokkaido UniversitySapporoJapan

Personalised recommendations